Presentation Description

Presented by Dr. Scott Coonrod at the Alliance for Contraception in Cats & Dogs’ 4th International Symposium on Non-Surgical Contraceptive Methods of Pet Population Control, April 8-10, 2010, in Dallas, Texas, U.S.

Presentation Transcript

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Spike Trish Coonrod

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Immunocontraception – a reversible birth control method that uses the body's immune response to prevent pregnancy
Immmunosterilization – Use of immune system to permanently sterilize animal.
How does immunocontraception/immunosterilization work? Molecules from the reproductive axis that are essential for fertility are identified
Recombinant (synthetic) forms of these molecules are produced using bacterial or eukaryotic systems
Inject reproductive antigen into host animal to generate an immune reaction that either neutralizes antigen or removes cell type that produces the antigen

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Antibody-based T-cell based Two main immunological approaches to removing fertility antigens from animal to induce contraception. Antibody removes target antigen (and possibly associated cells) by binding to the molecule rendering it functionally inactive. This technique can potentially be reversible once antibody titers go down T-cells (such as Macrophages)
attack and destroy cells containing
target antigen. Technique can be
permanent. Immune response can be pushed toward antibody (B-cell) or
T-cell based response depending on how the antigen is presented to the
immune system.

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X Hypothalamus X X X Generation of T-cell mediated response Good Not Good

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Cellular Response Hypo X X GnRH Antibody Response Ideal immunosterilant would be one that targets GnRH with antibodies (so you don't ablate the hypothalamus) and also targets the ovary with a T cell response (so you do ablate the germ cells).

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How would such a immunosterilant be produced
State of the art method for targeting GnRH will be described
in next talk
Here I will focus on some strong candidate ovarian antigens
for T cell targeting and also discuss how that antigens could be delivered.

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Tolerance, Regulatory T-cells (T Regs), and Autoimmunity Tolerance - The ability of the immune system to distinguish self from non-self and not mount a response to self antigens. Autoreactive T cells are deleted and autoreactive T cells that escape deletion are controlled by Tregs
Regulatory T-cells - Tregs prevent immune responses from becoming too strong by “training” immune system to tolerate self antigens.
Autoimmunity – Induced by an overactive immune response which mistakes certain self molecules as foreign pathogens. Tregs are “overwhelmed” by response and cannot prevent attack of tissues expressing these self antigens by the immune system.
During autoimmune response, proinflammatory T cells , macrophages, and proinflammatory cytokines (TNF, IL-1B, GM-CSF) can be activated leading to recruitment of inflammatory cells to self antigen expression tissues leading to tissue destruction.

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What is the cause of this potent autoimmune response. Removal of thymus gland at day 3 also removed resident Treg cells thus making female mice incapable of suppressing autoimmune attack when females became sexually mature.
d3tx phenotype caused by endogenous antigen(s) required for generation and maintenance of the ongoing autoimmune response.
Endogenous antigens that induce this potent germ-cell depleting immune response would likely make ideal contraceptive vaccine antigens.

MATER and PADI6-null two-cell developmental arrest likely due to failure to activate embryonic genome. MII GV MII Ovary PADI6 and MATER-null arrest PADI6 MATER and PADI6 are part of the same
complex in mouse oocytes

Production of CPV virus-like particles (VLPs) :

Production of CPV virus-like particles (VLPs) Contains only the VP2 capsid protein
Can be generated in mammalian or insect cells
Can bind and enter permissive cells (but are non-infectious)
Have similar stability and morphology to complete viruses w/o pathology Singh, P. et al. J Nanobiotech. 2006 Yuan and Parrish, Virology 2001

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The CPV-derived VLPs present two opportunities to generate enhanced immune responses.
They are multivalent carriers: An incorporated antigen can be displayed as 60 copies/capsid with spacing sufficient to cross-link receptors on lymphocytes, thus effectively inducing activation.
Most dogs and cats of reproductive age have been exposed to CPV/FPV, thus existing immune surveillance system will mount a classical immunological memory response to the immunosterilant vaccine. Therefore, even though the specific attached antigen may not have been the target of an immune response before, this still results in an enhanced immune response.

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(Courtesy of C. Parrish) VLP containing GnRH and MATER epitopes. Model of VLP structure– GnRH and MATER peptides
could potentially be inserted at the loop sites. These
sites are antigenic and recognized by antibodies

Final Immunosterilant Vaccine :

Final Immunosterilant Vaccine A combination of GnRH-VPL and MATER-VLP
Would likely rapidly reduce GnRH levels, leading to reduced hormone levels and infertility
Would get slower cellular immune attack against MATER thus permanently eliminating oocytes and follicular cells

Acknowledgements :

What if a dog or cat has not been exposed to CPV? :

What if a dog or cat has not been exposed to CPV? Juvenile animals may not have been exposed to CPV infection or vaccination
For these, we may need to give a booster dose of vaccine after the initial dose
But ideally we would like a single-injection vaccine

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Dogs (Mahi Brown)
Immunization of bitches with crude and purified native pZP with CFA or Alum induced infertility, abnormal estrous cycles, follicular cysts and degenerating oocytes.
Recombinant ZP2 and ZP3
Mice
Studies by Tung showed that either B or T cell response can lead to either neutralizing response or oophoritis.
Dog
Immunization of female dogs with recombinant ZP2 and ZP3 conjugated to diphtheria toxin led to the generation of canine ZP-specific antibodies. All females that were immunized with ZP2 became pregnant while 3 of 4 females immunized with ZP3 did not conceive. Ovarian histology revealed inhibition of follicular development in treated animals
Several other studies using recombinant ZP proteins showed negative results.
Study in rabbits showed that while native porcine ZP induced infertility in rabbits when the porcine zona prep was deglycosylated the immunized females did not respond and were fertile. (Jones)

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GnRH GonaCon, synthetic GnRH bound to adjuvant
In swine and deer, infertility has lasted up to five years after a single treatment.
GonaCon induces GnRH antibodies in cats and a recent publication demonstrated the induction of GnRH antibodies in female dogs. However, problems with adjuvant mediated side effects.

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Ideal immunosterilant would be one that targets GnRH with an antibody response and the ovary with a T cell response

Previous Research :

Previous Research

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Summary
Results with GnRH suggest that, once optimized, immunization with this reproductive antigen could generate a long term B-cell mediated immunocontraceptive response (more later on this from Dr. Schiller)
Results with native zona pellucida preparations provide a proof of principle that immunization of animals with this reproductive antigen can generate both a B and T cell mediated response neutralizing the zona pellicida and also stimulating follicular degeneration /oophoritis
However, results from zona deglycosylation and recombinant zona studies suggest that carbohydrate configuration is important for immunoreactivity.

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The prospect of an immunological approach to contraception that would disrupt the process of fertilisation itself has resulted in a considerable interest into research in this area. It has been known for some time that antibodies raised against the zona pellucida (ZP) can suppress fertility very effectively. However, the initial optimism of this approach has been marred by the appearance of an ovarian pathology characterized by disruption of folliculogenesis and depletion of the primordial follicle pool. Adverse auto-immune reactions have been observed in the ovaries of mice after the induction of immunity with mouse ZP3 epitopes. However, this was associated with lymphocytic infiltration of the ovarian stroma, which could be circumvented by careful selection of B-cell epitopes to induce reversible infertility. In order to identify similar epitopes on primate ZP3, epitope-mapping studies were performed and incorporated into chimeric vaccines that included a promiscuous T-helper cell epitope. Both single and triple peptide vaccines have been evaluated in vivo and no detrimental effects on ovarian function were observed. The resulting high titre antibodies bound exclusively to the ZP of marmoset and human ovarian sections and could suppress in vitro human sperm-egg binding by approximately 60%, but did not prevent pregnancy in actively immunised female marmosets. Thus, considerable research is still required to identify a combination of ZP3 epitopes that will induce infertility free of any unwanted side effects. Immunocontraception with Zona pellucida Proteins: Review Aitken, Cells Tissue Organs 166 Human Immunocontraception Research: Persistent problem with the immune system becoming “overstimulated” generating a T-cell mediated inflammatory response targeting ovary.
What's bad for people may be good for companion animal sterilization!!